Mycobacterium tuberculosis (MTb) is the causative agent of tuberculosis, which is responsible for nearly 2 million deaths per year worldwide. This figure is a compelling reminder that new avenues for tuberculosis vaccination and treatment are urgently needed, particularly in developing countries. MTb possesses an unusual cell wall that not only imparts drug resistance but also contains an abundance of trehalose-based glycolipids that are intimately involved in disease pathogenesis. The broad objective of the proposed work is to develop a chemical reporter strategy to probe trehalose-containing metabolites (TCMs) in MTb. This goal will be accomplished through three Specific Aims: (1) Synthesize azide-modified trehalose analogs;(2) evaluate azido trehalose analogs for metabolic incorporation into surface TCMs;(3) subject the azide-labeled TCMs to visualization and metabolomic analysis. Based on the current understanding of trehalose metabolism by MTb, we hypothesize that the proposed azido trehalose analogs will be metabolically incorporated into surface-exposed TCMs by the cell's own biosynthetic machinery. Subsequently, TCMs bearing the bioorthogonal azide reporter tag are expected to react with exogenously delivered probes outfitted with a mutually reactive functional group such as a cyclooctyne. The probe will also be equipped with either a fluorophore or affinity tag to permit detection or isolation, respectively. This strategy will provide a platform for visualizing and profiling the surface glycolipids of MTb, through which numerous applications can be envisioned, including imaging of wild-type MTb in cell and live animal models, evaluating changes in TCM expression during infection, and identifying new TCMs via metabolomic analysis. PUBLIC HEALTH RELEVANCE: The research described in this application will provide a chemical approach to exploring a unique class of molecules that are present on the cell surface of Mycobacterium tuberculosis (MTb), the causative agent of tuberculosis. These compounds, termed trehalose-containing glycolipids, are intimately involved in disease pathogenesis, which makes their study an important avenue for unraveling host-pathogen interactions. By developing a platform for imaging and profiling trehalose-containing glycolipids in MTb, new insights into tuberculosis therapeutic development may be discovered.